BACKGROUND AND
OBJECTIVES: In order to prolong the action and reduce systemic toxicity,
formulations of local anesthetic (LA) complexed with cyclodextrins (CD) have
been developed. This study determined the physical-chemical characterization
and evaluated the effects of inclusion complexes of racemic bupivacaine (S50-R50)
and 50% enantiomeric excess (S75-R25) bupivacaine with hydroxypropil-beta-cyclodextrin
(HP-b-CD) in rats, and comparing them with the solutions
currently used in the clinical practice.METHODS: Inclusion complexation of S75-R25 with HP-b-CD
(equimolar ratio 1:1) was characterized by phase-solubility studies varying
the concentrations of HP-b-CD and the temperature.
Affinity constants (K) for HP-b-CD and the thermodynamic
parameters for complexation were determined. Motor and sensitive anesthesias
were evaluated through the subarachnoid administration of the formulations in
the concentration of 0.5%.RESULTS: Inclusion complexation was observed through the increase in
aqueous solubility of LA in different temperatures and concentrations of HP-b-CD.
The in vivo tests demonstrated that S50-R50HP-b-CD
and S75-R25HP-b-CD reduced
latency (p < 0.001) without changing the recovery time of the motor block,
time for maximal effect, and total effect of the drugs. Besides, both formulations
increased the intensity (1.5 times, p < 0.001) and prolonged the duration
of analgesia compared to the free drugs.CONCLUSIONS: The complexes S50-R50HP-b-CD
and S75-R25HP-b-CD potentiated
the differential nervous block, and can be used to reduce the frequency of administration
or the dose of the LA to induce the same effect. The formulation containing
enantiomeric excess (S75-R25) bupivacaine showed to be interesting in the development
of safer formulations, and useful for the treatment of acute pain in the postoperative
period.

The advances in
basic and clinical research have expanded the pharmacological options for pain
treatment and local anesthetics (LA), which have the ability to block the excitation-conduction
process in peripheral nerves, are among the several classes of drugs used to
alleviate or eliminate pain. Even though bupivacaine has a great clinical applicability,
ropivacaine, levobupivacaine, and, more recently, the 50% enantiomeric excess
(S75-R25) bupivacaine have become new options for prolonged regional anesthesia.
However, the LAs currently used present limitations due to their relatively
short action and their toxicity for the central nervous and cardiovascular systems
1,2.

Aiming at prolonging
the duration of action and reducing systemic toxicity of these drugs, several
formulations of LA complexed with cyclodextrins (CD), cyclic oligosaccharides
resulting from the enzymatic hydrolysis of starch 3,4, have been
developed. Besides the physical-chemical stabilization, the main effects of
complexation with CD are observed especially in the change in aqueous solubility,
the liberation ratio, and pharmacokinetics and pharmacodynamic properties, modifying
the duration and intensity of the pharmacological effects of these drugs 5,6.

Animal studies
indicate that complexation with beta-cyclodextrins (b-CD) and some of its derivatives,
such as hydroxypropil-beta-cyclodextrin (HP-b-CD) (Figure
1), increases aqueous solubility and the duration of the nervous block induced
by racemic bupivacaine 7-11. Furthermore, a recent study demonstrated
that complexation of racemic bupivacaine (S50-R50) and the 50% enantiomeric
excess (S75-R25) with HP-b-CD potentiated the intensity and duration of analgesia
after sciatic nerve block in mice, being potentially advantageous for the relief
of postoperative pain 12.

Since bupivacaine
(S75-R25) is less toxic that racemic bupivacaine (S50-R50), the objective of
this study was to determine the physical-chemical characteristics and the effects
of the spinal block administration in rats of the inclusion complexation of
racemic bupivacaine (S50-R50) and enantiomeric mixture (S75-R25) with HP-b-CD,
and comparing them with the free solutions currently used.

METHODS

The solid complexes
were prepared mixing proper doses of HP-b-CD and S75-R25 in water in a molar
ratio of 1:1. Samples were agitated for 24 hours at room temperature. Afterwards,
the solution was lyophilized and stored at 20° C for later use. Enough doses
of the solid complexes were weighed to obtain the proper concentrations and
dissolved, afterwards, in a buffer (20 mM of HEPES with pH of 7.4 with 154 mM
of NaCl). The physical-chemical complex was characterized through solubility
studies according to the method described in the literature 13. Excess
local anesthetic (15 mM) was added to the buffer solution, 50 mM with pH of
7.4, in the presence of increasing doses of HP-b-CD (0, 5, 10, 20, 25, 30, 35
nM).

Solutions were
agitated at 110 rpm at different temperatures (10°, 15°, 20°, 30°,
and 40° C) until the system reached an equilibrium (24 h), and the concentration
of the anesthetic dissolved was determined by spectrophotometry (molar absorbance,
e = 470) 14. With these data, we obtained
the solubility isotherms, and the affinity constants (K) were measured at different
temperatures using equation 1, where: So is the initial concentration of the
LA in solution.

A van't Hoff chart
was built using the affinity constant (K) against the temperature and measuring
the thermodynamic parameters for the complex in question: enthalpy (DH°)
and entropy (DS°), according to equation 2, where
T is the temperature (in degrees Kelvin) and R is the ideal gas constant (8.31
kJ/mol) 15-17.

This experimental
study was conducted after approval by the Ethics Committee on Animal Experiments
of the Instituto de Biologia of the Universidade Estadual de Campinas (CEEA)
 IB  UNICAMP, which follows the rules of the Colégio Brasileiro
de Experimentação Animal (COBEA). Groups of five male, albino
rats, of the Wistar lineage, approximately 90 days old, weight varying from
200 to 250 g, were used. The animals came from the Centro de Bioterismo of UNICAMP
(CEMIB) and were exposed to light/dark, 12-hour cycles, with free access to
food and water, housed in groups (5 animals per cage), and acclimated to the
local of the experiment for at least 7 days.

The animals were
firmly contained while a needle connected to a Hamilton syringe was introduced
perpendicularly in the L5-L6 space (Figure
2) to perform the subarachnoid injection. The injection site was restricted
to the region where the spinal cord ends and the cauda equina begins, to reduce
the possibility of spinal lesion and to facilitate intervertebral access. When
the needle was inserted in the subarachnoid space, there was a sudden movement
of the tail. This reflex was considered an indication that the puncture was
successful. A maximun of 20 µL of the formulations was injected 18.

Motor and sensitive
blocks were evaluated simultaneously and every measurement was done by the same
investigator. Animals were observed for 24 h after the treatment to determine
the incidence of systemic (seizures and deaths) or local (failure to recover
normal movements) toxic effects. Experimental groups were characterized as follows:

The presence of
motor block was determined by "dragging" the hind legs and/or "closing" of the
fingers 19, and its intensity was evaluated according to the following
scores: 0 (normal use of the hind legs), 1 (unable to flexion completely the
hind legs), and 2 (cannot use the hind legs). Animals were evaluated every minute
for the first 5 minutes after the subarachnoid administration, followed by evaluation
at 10-minute intervals until complete recovery of the movements (observation
for at least 1 hour). Parameters evaluated were latency (interval between the
injection and development of motor block grade 1 or 2), length of time to reach
maximum score (Tmax), length of time to recover motor function, and
total effect of the LA (estimated by the area under the effect-time curve, AUC).

Sensitive block
was evaluated by a mechanical stimulus 20. A device called analgesimeter,
which generates a gradual increase in the strength (in grams) used by a plastic
extremity over the dorsal surface of the animal's paw, was used. To avoid the
analgesia produced by stress, each animal was wrapped in a towel, except for
its head and legs. Paw withdrawal reflex was considered an indication of the
pain threshold  Pain Withdrawal Threshold to Pressure (PWTP). Basal PWTP
was measured before the experiment and animals with a pain threshold below or
above 90 to 140 g were excluded, and a maximal pressure (cut-off) of 350 g,
considered as representative of a sensitive block, was established. After the
administration of the excipient or formulations, measurements were done for
five hours; at 15-minute intervals for the 1st hour, 30-minute intervals
for the 2nd and 3rd hours, and 60-minute intervals in
the last two hours. Analgesia was defined as an increase in the pressure threshold
of the animals at least 50% greater than in the group treated with the excipient.
The end-point of analgesia was established when there were no statistical differences
among the test and control groups 12.

The Mann-Whitney
test was used for the statistical analysis of the motor block (latency, Tmax,
recovery time, and AUC) and the data expressed as median (minimal and maximal
limits). PWTP values during sensitive blocks were expressed as mean ±
SD and the individual times were evaluated by the Variance Analysis (One-way
ANOVA) and posteriorly by the Tukey-Kramer test 21. A value was considered
statistically significant when p < 0.05.

RESULTS

It was possible
to observe, through the solubility phase tests, the complexation of S75-R25
with HP-b-CD at a pH of 7.4 in different temperature (Figure
3). The solubility diagram of the phase systems indicates formation of soluble
complexes with a linear increase in solubility of S75-R25 against the concentrations
of HP-b-CD tested in different temperatures.

Using linear regressions
obtained with the curves in figure 3 and equation 1,
the affinity constant (K) between S75-R25 and HP-b-CD in different temperatures
was calculated (Table I). The data obtained demonstrated
that elevating the temperature increases the values of the affinity constant
(K) due to an increase in the solubility of the drug, dislocating the equilibrium
to form the complex.

A van't Hoff curve
(Figure 4) was obtained from the temperature and affinity
constant (K) values using equation 2, obtaining, therefore, the thermodynamic
parameters of the complexation of S75-R25 with HP-b-CD.

Thermodynamic parameters
were obtained from figure 4, through linear regression
of the data. Values obtained for the complexation of S75-R25 with HP-b-CD
were: 21.86 kJ/mol of enthalpy (DH°), 90.40 J/mol/K
of entropy (DS°), and 5.07 kJ/mol of free energy
in the system (DS°). The analysis of this data
demonstrates that complexation between S75-R25 and the internal cavity of CD
is favorable, since the free energy of the system is negative, and the entropy
of the system is decisive for complexation of the drug with the CD.

Evaluation of
the Motor Block

When the anesthetic
solutions, free and in complexation, are administered by the subarachnoid route,
they had the same effect on the animal's motor function. However, the administration
of the excipient, HP-b-CD, did not produce any signs of motor block.

Comparing the experimental
groups individually, we observed that the latency of the motor block was significantly
reduced (p < 0.001) by the treatment with S50-R50HP-b-CD and S75-R25HP-b-CD
when compared with the free solution. However, other parameters, such as length
of recovery from the motor block, length of time for maximum effect, and total
effect of the drug did not change with complexation with HP-b-CD (Table
II).

Evaluation of
the Sensitive Block

The evaluation
of the sensitive block, through the PWTP, showed the effect-time relationships
for the free and complexed LA. The effects of the excipient (HP-b-CD), free
drugs (S50-R50 and S75-R25), and inclusion complexation (S50-R50HP-b-CD
and S75-R25HP-b-CD) administered by the subarachnoid route, were
evaluated (Figure 5 and 6).

The treatment with
S50-R50HP-b-CD increased the nociceptive
threshold when compared to the free drug, at all times of the experiment. In
the initial phase (up to 180 minutes), i.e., the end point of the analgesia
induced by free S50-R50, there was a significant increase (p < 0.001) in
the intensity of the analgesia (approximately 1.5 times more than the free S50-R50)
(Figure 5).

The inclusion complex,
S75-R25HP-b-CD, also changed the answer of the animals to nociceptive
stimuli, increased pain threshold and the duration of the antinociceptive action
up to 5 hours after its administration (Figure 6). The
intensity of the analgesia increased (1.4 times) when compared with the free
S75-R25 after 180 minutes of treatment (p < 0.001).

Both systems of
controlled release (S50-R50HP-b-CD and S75-R25HP-b-CD)
increased the intensity and prolonged the duration of the analgesia, and the
effects of the complexes was practically stable during the last two hours (Figures
5 and 6), demonstrating they are more efficient than
the free S50-R50 and S75-R25.

DISCUSSION

The ability of
the CDs to form inclusion complexes depends, essentially, on the compatibility
of the esters and the polarity between the CD and complexed molecule. Moreover,
the forces that drive the complexation have been attributed to the high energy
of the repulsion forces of the water present in the cavity of the CDs, the van
der Waals interactions, hydrogen bonds, and hydrophobic interactions 22,23.
Complexed molecules remain oriented in the direction of the greatest contact
between its hydrophobic portion and the nonpolar cavity of the CD, while its
hydrophilic portion is in contact with the hydroxyl groups of the CD or exposed
to the aqueous environment 3,23. In fact, the results of the physical-chemical
characterization tests indicate that S75-R25 interacts with the cavity in the
molecule of HP-b-CD, and that this interaction increases the aqueous solubility
of the LA, being energetically favorable, and indicating a possible increase
in the bioavailability of the drug.

For comparison,
the groups treated with S50-R50 and S50-R50HP-b-CD were included
in the tests that evaluated the anesthetic efficacy. Although complexed formulations
did not change the duration or the intensity of the motor block, there was a
reduction in the latency time, and a potentiating effect on the differential
nervous block induced by the LA. These results agree with those obtained previously
with the sciatic nervous block in mice, indicating that complexation with HP-b-CD
reduces the length of time to establish motor anesthesia, promoting a faster
beginning of action without prolonging or intensifying its duration 12.

The ideal LA should
be long acting, have low toxicity, and greater selectivity for sensitive than
motor fibers, since this characteristic of maintaining analgesia without affecting
excessively motor function has great clinical relevance 24.

Evaluation of the
sensitive block induced by the subarachnoid injection of inclusion complexes,
S50-R50HP-b-CD and S75-R25HP-b-CD, demonstrates
an increase in the intensity and duration of the analgesia when compared to
the free drugs. The potentiation of the analgesic effect promoted by the complexation
with CD may be connected to the increase in the total concentration of the LA
available with these formulations, as well as by the formation of a barrier
that hinders their binding to liquor proteins and being captured by the blood,
since the free drug has a large capacity of binding to liquor proteins, therefore
reducing the amount of the drug that is available.

When lipophilic
drugs (such as S50-R50 and S75-R25) are administered by the subarachnoid route,
they are quickly distributed in the cerebrospinal fluid. Consequently, their
onset of action is faster, but the duration of their effect is shorter 25.
Thus, if complexation with CD makes a greater fraction of the LA available while,
at the same time, increasing the length of time it is in contact with the nerve,
complexed drugs would be of great clinical utility for potentiating is analgesic
action.

Even though both
complexes, S50-R50HP-b-CD and S75-R25HP-b-CD, enhanced
the differential nervous block, the formulation with 50% enantiomeric excess
(S75-R25) bupivacaine is better than racemic bupivacaine (S50-R50) due to its
lower toxic potential, favoring the development of safer formulations that are
more clinically useful for the treatment of postoperative acute pain, while
being administered in smaller doses.

The results of
this study demonstrated that it is possible, through nano-structure formulations
especially designed for each LA, to increase the differential nervous block
and the total duration of the anesthesia after the administration of racemic
bupivacaine (S50-R50) and 50% enantiomeric excess (S75-R25) bupivacaine. Thus,
the proposed non-emulsion formulations of LAs can be used to decrease the frequency
of the administration or the dose necessary to induce the same effect, which
is interesting for long acting drugs with high systemic toxicity, such as bupivacaine.

14. de Paula E, Schreier S  Use of a novel method for determination of partition coefficients to compare the effect of local anesthetics on membrane structure. Biochim Biophys Acta, 1995;1240:25-33. [ Links ]